Functional expression of a two-transmembrane HtrII protein using cell-free synthesis.
Functional expression of a two-transmembrane HtrII protein using cell-free synthesis.
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DOI：
10.2142/biophysics.7.51
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Kojima C
中科院分区：
文献类型：
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作者：
Sudo Y;Tanaka R;Kobayashi T;Kamo N;Kohno T;Kojima C
为实现跨膜蛋白的功能表达，提出了一种无需折叠的无细胞合成方法。通过添加不同的大分子可溶性标签(硫氧还蛋白、谷胱甘肽S转移酶、绿色荧光蛋白和麦芽糖结合蛋白)，翻译了法老细菌转导蛋白PHtrII的跨膜区。在这个系统中，所有的融合PHtrII都被翻译成一个可溶的部分，可能形成了巨大的胶束状结构。加入去污剂n-十二烷基-β-d-麦芽糖苷，以增强PHtrII疏水区的增溶作用。利用PHtrII与膜上的法老恐视紫质(PPR)形成信号复合体这一事实，以及在洗涤剂存在的情况下，利用下拉试验检查表达的具有不同标签的PHtrII的活性。所有标记的pHtrII都显示了与PPR的结合活性。有趣的是，与PPR的结合活性与可溶性标签的相对分子质量呈正相关。因此，更大的可溶标签会导致更高的结合活性。我们可以证明，我们的方法有利于活性膜蛋白的制备，也可能适用于更大的膜蛋白，如7-跨膜蛋白。
An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-d-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.